Changes in the Major Odorants of Grape Juice during Manufacturing of Dornfelder Red Wine

Application of the aroma extract dilution analysis (AEDA) on a distillate prepared from freshly squeezed juice of Dornfelder grapes revealed (3Z)-hex-3-enal and trans-4,5-epoxy-(2E)-dec-2-enal with the highest flavor dilution (FD) factors. In contrast, in the final Dornfelder wine prepared thereof, the highest FD factors were found for 2-phenylethyl acetate, 2-phenylethan-1-ol, and (E)-β-damascenone. However, for example, among others, (3Z)-hex-3-enal no longer appeared as an important odorant. To monitor the olfactory changes occurring in single processing steps from Dornfelder grapes to the final wine, selected odorants in grape juice, must, and young as well as aged wine from the same batch of Dornfelder grapes were quantitated. In particular, (3Z)-hex-3-enal and hexanal decreased considerably during mashing, while, as to be expected, the concentrations of yeast metabolites, e.g., odor-active alcohols and esters, drastically increased during fermentation. To reveal the influence of barrel aging, the odorants of the same Dornfelder wine aged in either barrique barrels or steel tanks were compared.


■ INTRODUCTION
−7 The results showed that, in particular, the grape variety, the fermentation process, and the storage in barrels are key parameters influencing the overall aroma of the final wine.
Several studies have already been undertaken to clarify the odorants of different grape varieties.Beak et al. 8 and Fan et al. 9 analyzed the odorants in grapes of the varieties Muscadine, 8 Cabernet Gernischt, 9 Cabernet Sauvignon, 9 Cabernet Franc, 9 and Merlot 9 by gas chromatography−olfactometry (GC−O).Unexpectedly, Fan et al. 9 identified the same odor-active compounds in all examined grape varieties, but their concentrations varied, thereby indicating that the characteristic odors of the different varietals depended upon quantitative rather than qualitative differences in the odor-active compounds.
In particular, the well-known amino acid metabolism, known as the Ehrlich degradation, leads to the formation of a certain group of potent odorants in many alcoholic beverages, such as wine, e.g., alcohols, aldehydes, and esters. 4,10,11Hernańdez-Orte et al. 12 added selected amino acids to grape juice, for example, phenylalanine, and observed that the content of 2-phenylethan-1-ol was higher in the fermented grape juice supplemented with the amino acid than in the grape juice without the addition.Besides the amino acid metabolism, further odorants present in the final wine were either transferred directly from the grape juice or were formed from odorless precursors in grapes, such as glycosides and S-conjugates.The hydrolysis of glycosides can occur either enzymatically during fermentation or by acid hydrolysis during aging.Ugliano and Moio 13 found that the yeast-driven enzymatic hydrolysis of glycosides was the major formation pathway for linalool and geraniol, while an acid hydrolysis led, among other compounds, to the generation of terpinen-4-ol and (E)-β-damascenone.In a last step of winemaking, storage in oak barrels has a huge influence on the aroma of the wine, 4 and wines stored in oak, especially in barrique barrels, are commonly rated by the consumer to be of higher quality.For example, Jarauta et al. 14 compared the volatiles of red wine that was stored in oak barrels to the same wine stored in stainless-steel tanks.Aging in oak affected many volatiles, including (4R,5R)-5-butyl-4-methyloxolan-2-one (whiskey lactone and oak lactone) and 4-hydroxy-3-methoxybenzaldehyde (vanillin), which are considered as key oakderived compounds. 4o summarize, numerous studies have been published on the odor-active compounds of red wine, and also, the influence of single manufacturing steps on changes in wine volatiles has been reported.However, to the best of our knowledge, no data are available on changes of important odorants in Dornfelder grape juice on the way from grape juice to the final wine by application of the sensomics concept for the identification of odorants. 15ost previous studies were focused on either one single step of the manufacturing process or only a few odorants.Therefore, the aim of the present study was, first, to characterize the odorants in a freshly squeezed Dornfelder grape juice and in a steel tankaged wine produced thereof.Second, monitoring the concentration of selected odorants during wine production using the same batch of grapes should be performed, and finally, major odorants in red wine of the same vintage and vineyard either stored in barrique barrels and steel tanks should be compared to elucidate the influence of the oak material on the odorant spectrum.

■ MATERIALS AND METHODS
Samples.Dornfelder samples were obtained from a wine grower in the Rheinhessen region (Germany).Grape juice odorants were analyzed between 1 and 4 days after harvest of the fruits.For mash preparation, grapes were pressed, the mash was kept for 24 h, and a nonyeasted must was received after pressing.For wine preparation, Saccharomyces cerevisiae yeast was added to the non-pasteurized mash, and the material was fermented for 2 weeks.The fermented mash was pressed to obtain a young wine, of which one half was directly analyzed.The second half was filled in used French oak barrels and stored for 7 months.Another batch of young Dornfelder wine from the same grapes was stored in either steel tanks for 6 months or barrique barrels for 17 months.The barrique barrels (225 L) were made of French oak, and the barrels had already been used twice.Storage took place in a dark cellar at an average of 12 °C.
For compound quantitation, either a one-dimensional GC−MS system or a two-dimensional heart-cut GC−GC−MS system was used.As a one-dimensional instrument, a Varian CP 3800 gas chromatograph (Darmstadt, Germany) equipped with a CTC Analytics Combi PAL autosampler (Zwingen, Switzerland) was connected to a Varian Saturn 2000 mass spectrometer operated in the chemical ionization (MS−CI) mode with methanol as the reagent gas.The Agilent DB-FFAP column, 30 m × 0.25 mm inner diameter, 0.25 μm film, was operated as described above.The injection volume was 2 μL.The Varian MS Workstation software was used for the evaluation of the mass spectra.As a two-dimensional heart-cut instrument, a GC−GC−MS system with a Thermo Trace GC Ultra gas chromatograph equipped with a CTC Analytics Combi PAL autosampler was coupled to a Varian CP 3800 as the second gas chromatograph.The fused silica column in the first gas chromatograph was the Agilent DB-FFAP column, 30 m × 0.32 mm inner diameter, 0.25 μm film, as described above, and an Agilent DB-1701 column, 30 m × 0.25 mm inner diameter, 0.25 μm film, was installed in the second gas chromatograph.The column end in the first gas chromatograph was connected to a Thermo moving column stream switching (MCSS) device, and the column end in the second gas chromatograph was connected to a Varian Saturn 2200 mass spectrometer operated in the MS−CI mode with methanol as the reagent gas.The oven temperature programs were comparable, as mentioned above.The injection volume was 2 μL.The Varian MS Workstation software was used for the evaluation of the mass spectra.
Isolation of Volatiles.The grape juice was obtained by means of a Philips Viva Collection, HR 1832/00 kitchen squeezer (Hamburg, Germany).Immediately after squeezing, an aqueous saturated calcium chloride solution (100 mL) was added to avoid enzymatic reactions.Volatiles were isolated by extraction with diethyl ether followed by application of the solvent-assisted flavor evaporation (SAFE). 28The distillate was concentrated to 1 mL.The detailed workup procedure applied to all samples was performed as previously described for red wine. 17roma Extract Dilution Analysis (AEDA).The concentrated volatile fractions were stepwise-diluted 1:2 with diethyl ether, and each diluted sample was subjected to GC−O.Each odorant was assigned a flavor dilution (FD) factor, representing the dilution factor of the highest diluted sample in which the odorant was detected during GC− O analysis.The analysis was carried out as previously described. 17dorant Quantitation.Various amounts of the respective sample (0.05−500 mL) were used depending upon the amounts of the target ) and the respective internal standards were calculated from the extracted ion chromatograms using the quantifier ions detailed in Table 1.The concentration of each target compound was then calculated from the area counts of the analyte peak, the area counts of the standard peak, the amount of Dornfelder sample used, and the amount of standard added, by employing a calibration line equation (Table 1).To obtain the calibration line equation, solutions of the reference analyte and standard were mixed in different concentration ratios and analyzed under the same conditions followed by linear regression.Detailed information, e.g., on quantifier ions, for compounds 2, 4−5, 7, 11, 13, 16−17, 22−24, 28−32, 34−36, 38, 40, and 42−44 are given in the previous publication. 17ompound 1 was quantitated enzymatically using an ultraviolet (UV) test kit (R-Biopharm, Darmstadt, Germany).All odorants were consecutively numbered according to their retention time on the DB-FFAP column.b Each odorant was identified by comparing its retention indices on two fused silica columns of different polarity (DB-FFAP and DB-5), its mass spectrum obtained by GC−MS, as well as its odor quality perceived during GC−O to data obtained from authentic reference compounds analyzed under equal conditions.c Odor quality as perceived at the sniffing port during GC−O.d Retention index: calculated from the retention time of the compound and the retention times of adjacent n-alkanes by linear interpolation.e Flavor dilution factor: dilution factor of the highest diluted sample prepared from the concentrated volatile fraction in which the odorant was detected during GC−O by three panelists.f An unequivocal mass spectrum of the compound could not be obtained; identification was based on the remaining criteria detailed in footnote b.All odorants were consecutively numbered according to their retention time on the DB-FFAP column.b Means of 2−3 repetitions; standard deviations were ≤15%.d The odor activity value was calculated as ratio of the concentration to the odor threshold concentration.e Concentration refers to a Dornfelder grape juice of the consecutive year.f Odor threshold concentration of the racemate.All odorants were consecutively numbered according to their retention time on the DB-FFAP column.b Each odorant was identified by comparing its retention indices on two fused silica columns of different polarity (DB-FFAP and DB-5), its mass spectrum obtained by GC−MS, as well as its odor quality perceived during GC−O to data obtained from authentic reference compounds analyzed under equal conditions.c Odor quality as perceived at the sniffing port during GC−O.d Retention index: calculated from the retention time of the compound and the retention times of adjacent n-alkanes by linear interpolation.e Flavor dilution factor: dilution factor of the highest diluted sample prepared from the concentrated volatile fraction in which the odorant was detected during GC−O by three panelists.f These odorants were not separated on the fused silica column used for AEDA; the FD factor refers to the mixture.g An unequivocal mass spectrum of the compound could not be obtained; identification was based on the remaining criteria detailed in footnote b.
To obtain deeper insight into the role of single odorants in the overall olfactory profile, odorants with high FD factors (at least from FD of 64) were selected for quantitation using one-or twodimensional GC−MS systems.Stable isotopically substituted odorants were employed as internal standards.The results (Table 3) revealed concentrations ranging between very low concentrations of 0.087 μg/L for hex-1-en-3-one and high concentrations of 880 μg/L for hexanal, showing the highest amounts.The second highest concentration was determined for the second green, grassy smelling aldehyde (3Z)-hex-3-enal, followed by linalool and 4-hydroxy-3-methoxybenzaldehyde.

Changes in the Concentrations of Selected Odorants in Single Steps of the Manufacturing Process of a Dornfelder Wine.
A comparison of the odorants in the Dornfelder grape juice (Table 2) to those in the Dornfelder wine (Table 4) showed large differences.Therefore, to visualize the impact of single steps in the entire manufacturing process on the overall olfactory profile, selected odorants of different chemical classes were quantitated in a Dornfelder grape juice, must, and young and oak wood-aged wine, taken from the same batch of grapes.Volatiles from the individual samples were isolated and quantitated using stable isotopically substituted odorants as internal standards.The highest concentration in this batch of Dornfelder grape juice was detected for hexanal (1300 μg/L) (Table 5).The concentration of the second green, grassy smelling compound, (3Z)-hex-3-enal, was determined with 91 μg/L in the juice.Both aldehydes decreased during the manufacturing process, and while (3Z)-hex-3-enal was only detected in the juice, hexanal decreased by a factor of 20 during mashing but was no longer detectable in the wine.This was probably due to a reduction to hexan-1-ol during alcoholic fermentation. 36Besides hexanal and (3Z)-hex-3-enal, also the concentrations of linalool, ethyl 3-methylbutanoate, ethyl 2methylpropanoate, 2-methoxyphenol, and (E)-β-damascenone decreased during mashing, probably as a result of bioconversions caused by grape enzymes (Table 5).By destruction of the grape cells during mashing, the enzymes present in the cells are released and an enhanced enzyme reaction is possible.For example, Oliveira et al. 37 traced the decrease of C 6 -aldehydes to enzyme reactions, and Rapp et al. 38 attributed the decrease of ethyl esters to enzymatic reactions during mashing.During fermentation, the concentrations of 2-phenylethan-1-ol, linalool, ethyl 3-methylbutanoate, 2-methoxyphenol, (E)-β-damascenone, 3-hydroxy-4,5-dimethylfuran-2(5H)-one, 2-and 3-methylbutanoic acid, 2-and 3-methylbutan-1-ol, but also ethyl 2methylpropanoate increased (Table 5).2-Phenylethan-1-ol and 2-and 3-methylbutan-1-ol are well-known compounds formed by yeast metabolism and are undoubtedly formed by a degradation of the respective parent amino acids 2-phenylalanine, isoleucine, and leucine following the Ehrlich pathway.Figure 1 shows the influence of the manufacturing process of Dornfelder wine on the concentrations of odorants formed by yeast fermentation.Except for ethyl 2-methylpropanoate, which was predominately formed during aging, juice fermentation was the main step in the formation of the odor-active alcohols, ethyl esters, and acids.
Influence of Aging in Oak Barrels on Important Odorants in Dornfelder Red Wine.Dornfelder wine prepared from the same batch of grapes was aged in either barrique barrels or steel tanks, and 26 major odorants were quantitated (Table 6).The highest concentration was determined for acetic acid in barrique barrels (560 mg/L) compared to 270 mg/L in steel tanks.Similar data were found for 2-and 3-methylbutan-1-ol (350 mg/L, barrique; 290 mg/L, steel) and 2-phenylethan-1-ol (26 mg/L, barrique; 21 mg/L, steel).Nearly all quantitated odorants increased as a result of the oak wood contact.Only 2-phenylethyl acetate decreased.As to be expected, the only compound exclusively detected in the barrel aged wine was (4R,5R)-5-butyl-4-methyloxolan-2-one.Otsuka et al. 39 identified 4-[(3,4-dihydroxy-5-methoxybenzoyl)oxy]-3-methyloctanoic acid in oak wood and suggested it as the precursor of 5-butyl-4-methyloxolan-2-one.Masson et al. 40 later clarified the pathway for the formation of 5-butyl-4methyloxolan-2-one.This component may occur in four stereoisomers, but in oak wood, only two of them were previously identified. 41According to Garde-Cerdań and Anci ́n-Azpilicueta, 42 (4R,5R)-5-butyl-4-methyloxolan-2-one is regarded as the most important volatile of oak wood extracting into wine during barrel aging.A further difference in the odorants between the steel tank and the barrique barrel-aged wine was, for example, 3-(methylsulfanyl)propan-1-ol, which increased by a factor of more than 25 in the barrel wine (Table 6).Also, the amounts of 4-hydroxy-3-methoxybenzaldehyde, 4-ethylphenol, and 4-ethyl-2-methoxyphenol increased nearly 30 times during barrel aging.The higher concentrations of these three compounds in the barrique wine are suggested to be a result of lignin degradation, because the ring substitution of ferulic acid is common in all compounds.The pyrolysis of lignin is well-known during barrel toasting.Spillman et al. 43 confirmed that, in oak barrels, 4hydroxy-3-methoxybenzaldehyde was formed as a lignin degradation product, mainly during coopering.Chatonnet et al. 44 mentioned that 4-ethylphenol and 4-ethyl-2-methoxyphenol might have a microbiological origin.They supposed that these compounds were formed in wines during aging by some yeast species belonging to the genus Brettanomyces in the presence of hydroxycinnamic acid.
The study confirmed for the first time the molecular background of the huge olfactory changes occurring on the way from grape juice to the final Dornfelder red wine.Interestingly, of the selected quantitated odorants, only the concentrations of linalool and (E)-β-damascenone were identical in the grape juice and the final Dornfelder wine prepared thereof.However, the amounts of the free compounds present in the juice were degraded/lost during must preparation but were then released during fermentation/aging from their precursors in the juice.In general, the key steps in the formation of the overall olfactory profile of Dornfelder red wine are the significant reduction of the concentrations of both green, grassy smelling aldehydes delivered from the juice as well as the formation of yeast metabolites generated by amino acid degradation.Aging in a steel tank did not show differences in most of the major odorants of the Dornfelder red wine, but the steel tank wine consequently did not contain odorants released from the oak barrels, such as (4R,5R)-5-butyl-4-methyloxolan-2one.

Figure 1 .
Figure 1.Changes in the concentrations of selected alcohols, ethyl esters, and acids during manufacturing of Dornfelder red wine.Concentrations are given in Table5.Standard deviations were ≤12%.

Table 2 .
Twenty Two Important Odorants (FD Factor of ≥16) in the Volatile Fraction of Dornfelder Grape Juice

Table 4 .
Important Odorants (FD Factor of ≥16) in the Volatile Fraction of Dornfelder Wine Aged in Steel Tanks

Table 5 .
Concentrations a (μg/L) of Selected Odorants in Dornfelder Grape Juice, Must, and Young and Oak Wood-Aged Wine Taken from the Same Batch of Grapes a Means of 2−3 repetitions; standard deviations were ≤12%.b All odorants were consecutively numbered according to their retention time on the DB-FFAP column.c Not detected during GC−O analysis.d These odorants were not separated on the fused silica column used for quantitation; the concentration refers to the mixture.

Table 6 .
Concentrations a (μg/L) of Odorants in Dornfelder Red Wine Aged in Either Barrique Barrels (BBA) or Steel Tanks (STA) a Means of 2−3 repetitions; standard deviations were ≤16%.b All odorants were consecutively numbered according to their retention time on the DB-FFAP column.c Not detected during GC−O analysis.d These odorants were not separated on the fused silica column used for quantitation; the concentration refers to the mixture.